Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multi-Drug Resistance pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multi-Drug Resistance pumps (SMRs) are 100-110 residue, dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. Unfortunately, the conformational plasticity that enables these proteins to transport such diverse substrates has led to very different structural models and structural interpretations, particularly for the protein in detergent micelles. Thus we propose to determine the lipid bilayer-embedded structures of the conformations making up the functional cycle of the SMR from Staph aureus that provides resistance against common disinfectants, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Developing a high performance discoidal peptide-lipid bilayer nanodiskette system for NMR studies of membrane proteins, 2) Establishing the relative topology for the Smr monomers (parallel vs inverted), followed by full structure determination of the apo-form of the protein in nanodiskettes, 3) Identifying the binding determinants for transportable drugs and inhibitors, 4) Defining the structural changes in the substrate-bound, and inhibitor-bound Smr transporter, and 5) Characterizing the fold of the most controversial family member, EmrE, in lipid nanodiskettes as a function of protonation state and TPP binding. PUBLIC HEALTH RELEVANCE: The Small Multidrug Resistance transporter protein protects strains of Staph aureus that carry it from common hospital disinfectants. We will determine the three dimensional structure of the protein by itself, and in complex with the compounds that it transports, in order to understand how it functions, and ultimately guide the design of inhibitors of the transporter.